Biological Systems and Formalizing Experimental Knowledge

Summary: BIOCHAM (the BIOCHemical Abstract Machine) is a software environment for modeling biochemical systems. It is based on two aspects  the analysis and simulation of boolean, kinetic and stochastic models and the formalization of biological properties in temporal logic. BIOCHAM provides tools and languages for describing protein networks with a simple and straightforward syntax, and for integrating biological properties into the model. It then becomes possible to analyze, query, verify and maintain the model with respect to those properties. For kinetic models, BIOCHAM can search for appropriate parameter values in order to reproduce a specific behavior observed in experiments and formalized in temporal logic. Coupled with other methods such as bifurcation diagrams, this search assists the modeler/biologist in the modeling process.

The Proof is in the Process

Celsus detailed the four cardinal signs of inflammation: rubor (redness), tumor (swelling), calor (heat) and dolor (pain). Inflammation is the body's way of fighting back. Celsus was right on target. Since then, we've learned that inflammation involves many tissues and myriad chemical mediators.Ruth D. Thornton, PhD, Chair and Professor, Biochemistry/Molecular Biology and others in the department are each studying a small piece of the very large puzzle that is inflammation.

"Of the major mediators, the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF), are among the first on the scene," explains Dr. Thornton. These cytokines act on many different cells close to an injury site, but they also can travel through the bloodstream to distant tissues.  Cytokines activate many other defensive reactions, including the production of nitric oxide and matrix metalloproteinases.

Usually, inflammation is acute and healing follows. Inflammation becomes chronic when IL-1 and TNF remain even after the initiator of inflammation has disappeared. These mediators help to establish chronic inflammation, such as that seen in rheumatoid arthritis (RA).

The researchers in the biochemistry/molecular biology department are studying what occurs just before, after and while inflammation takes hold in RA, using periodontitis as a model in some instances because it's easier to study. "Even though we can identify microbes as the cause of periodontitis, while the cause of RA is unknown, there are similarities; the nflammatory cytokines are systemic in both," notes Dr. Thornton. "What we learn from one should apply to the other, and hopefully to many other inflammatory diseases as well."

The researchers

Dr. Thornton is interested in learning what IL-1 does to activate synovial cells that line the joints of people with RA.  Farzaneh Daghigh, PhD, assistant professor, is studying the effects of nitric oxide, a soluble gas, on inflammatory disease.  Grzegorz Gorski, MD, PhD, instructor, is interested in which genes are slightly different (have polymorphisms) in RA patients from the genes of others without RA. Ruth Carter Borghaei, PhD, associate professor, is studying the effects of inflammation on gene expression.

Dr. Borghaei's work has been continually funded by the National Institutes of Health for the past seven years.  She's been taking a detailed look at the genes called matrix metalloproteinases collagenase-1 (MMP-1) and stromelysin (MMP-3). Both are significant because they are involved in normal physiological tissue remodeling as well as in a number of pathological processes, including periodontitis, RA, cancer, angiogenesis, atherosclerosis, emphysema and osteoporosis.

Dr. Borghaei's research focuses on identifying and studying mechanisms involved in transcriptional regulation of these genes in response to cytokines."The goal of my research is to identify transcription factors involved in regulating expression of MMP-1 and MMP-3 during inflammation," notes Dr. Borghaei. "We've found that a particular transcription factor, NFkB, which usually increases gene expression in response to inflammation, actually works as a repressor for MMP-3. So IL-1 increases MMP-3, but it also increases a factor that limits the increase in MMP-3.  That sounds complicated, but it's kind of like having an accelerator and a brake on at the same time," she explains.  "This finding is important, not only for our understanding of how the MMP-3 gene is regulated during inflammation, but it may also increase our understanding of gene regulatory mechanisms in general," adds Dr. Borghaei.

Dr. Gorski is interested in locating genetic differences between patients who have RA or other inflammatory diseases such as lupus (SLE - systemic lupus erythematosus) and those who do not have the disease. "These gene differences might, at least partially, explain an RA patient's inability to 'turn off' the cascading inflammatory process," notes Dr. Gorski. "It could also explain why some people have the disease while others don't." Another project of Dr. Borghaei's also examines a polymorphism in the MMP-3 gene that affects the ability of NFkB to repress MMP-3 production.

Dr. Daghigh is studying the effects of nitric oxide (NO), a free radical generated in biological systems. "NO functions at low levels as a signal in diverse physiological processes, such as blood pressure control, neurotransmission, learning, memory and many others," Dr. Daghigh explains. "Excessive NO generated from the enzyme inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of inflammatory diseases." Dr. Daghigh has been able to show that human gingival fibroblasts (from patients with periodontitis) are a source of NO. Recent results from her research indicate that cytokines stimulate iNOS to produce large amounts of NO. This work has been accepted for publication in the Journal of Periodontology.

Dr. Thornton has taken a wider view of inflammation by searching for genes that are stimulated by the proinflammatory cytokine IL-1. Among the many genes she found to be upregulated by IL-1, most, as

expected, participated in destructive functions. However, several also had constructive functions, such as bone morphogenetic protein (BMP-2), which helps to build bone, the opposite of MMP action. This work was done with Martin J. Fowler, DO '00, when he was a graduate student in the department. "My work is focused right now on hypoxia inducible factor-1 (HIF-1), a transcription factor which 'turns on' other genes potentially important in the inflammatory process," notes Dr. Thornton. "HIF-1, as its name suggests, is known to be controlled by hypoxia [when cells don't have enough oxygen], but this form of regulation by cytokines is new."

Are Dr. Thornton and other researchers in biochemistry/ molecular biology looking for a cure for RA and periodontitisBorghaei. "Some people were studying retroviruses for a time, then HIV happened and some of the pieces of the puzzle were already in place. Everyone wants to do research with the goal of a cure, but how things work has value in and of itself. It's about the process," she emphasizes, echoing a familiar theme in the department of biochemistry/molecular biology. "How do you hope to eventually arrive at a cure if you don't understand the process?"

Build DNA and Other Molecules

What Poison? Bacterium Uses Arsenic to Build DNA and Other Molecules

From elephants to the bacterium Escherichia coli, all forms of life on Earth depend on the same six elements: oxygen, carbon, hydrogen, nitrogen, phosphorus, and sulfur. “The paradigm is that the chemistry of life is so specific that any change in chemistry also changes molecular stability and reactivity, which would not be tolerated,” says Clara Chan, a geomicrobiologist at the University of Delaware, Newark.

In a paper published online by Science .1197258) this week, however, an exception to that rule makes a surprising debut. Meet GFAJ-1, a bacterial strain that researchers say can replace the phosphorus in its key biomolecules, including DNA, with the legendary poison arsenic. “This is a very impressive and exciting discovery,” says Barry Rosen, a biochemist at Florida International University in Miami. “The implication of this work is that life can be quite different from what we know,” agrees Chan.

In 2009, Felisa Wolfe-Simon, a geomicrobiologist based at the U.S. Geological Survey in Menlo Park, California, and two colleagues argued that arsenic could have stood in for phosphorus in ancient living systems. Phosphorus, in the form of the compound phosphate, forms the backbone of strands of DNA and RNA, as well as ATP and NAD, two molecules key to energy transfer in a cell. Arsenic, Wolfe-Simon pointed out, sits just below phosphorus on the periodic table and has similar chemical properties. Indeed, its toxicity to people and most forms of life arises when cells try to use arsenic in lieu of phosphorus.

Despite that, Wolfe-Simon speculated that some microbes might be able to adapt to using arsenic. Others were skeptical. The arsenic-containing compound arsenate is much more unstable than phosphate in water, and no cell would be able to cope with that, critics argued.

To test her hypothesis, Wolfe-Simon collected mud from Mono Lake, California, a desert body of water known for having high arsenic levels, and grew the microorganisms from it in increasing concentrations of arsenate. She didn't add any phosphate or other phosphorus-containing compounds to the growth medium, as is typically done to sustain microbes. Instead, she periodically transferred the growing cultures to a new dish to reduce the concentration of any original phosphorus to the point that any microbe making new DNA or other biomolecules would need to use the arsenic to survive. 

Like others, says Wolfe-Simon, she didn't really expect to find any survivors. So she was thrilled and surprised when one evening she checked the latest cultures under the microscope and saw fast-moving bacteria. She rechecked the components of the culture media to confirm there were no phosphorus contaminants. She and her colleagues then began to subject the microbes to sophisticated analyses to see if arsenic had been utilized by the bacteria. “I held my breath with every one,” says Wolfe-Simon.

One form of mass spectrometry showed that the arsenic was inside the bacterial cells and not some impurity sticking to the outside of the cell. When the researchers added radioactively labeled arsenate to the bacteria's culture, they were later able to discern its presence in the protein, lipid, nucleic acid, and metabolite fractions of the cells, suggesting that arsenic had been incorporated in molecules forming each fraction. They also separated out the DNA from the bacteria and analyzed its composition using a technique called high-resolution secondary ion mass spectrometry; the isolated DNA contained arsenic.

Tests utilizing the intense x-rays at a synchrotron facility offered additional support, indicating that at least some of the arsenic in the bacteria was in the form of arsenate with the appropriate molecular bonds to carbon and oxygen atoms to replace the phosphates in DNA and other molecules.

Such work has convinced many that Wolfe-Simon's team has isolated a bacterium that uses arsenic to grow. “The organization of the experiments presents convincing and exhaustive results,” says Milva Pepi, an environmental microbiologist at the University of Siena in Italy. But not everyone agrees. Rosen finds the study “believable” but says he still has lingering concerns that the arsenic is simply concentrated in the bacterial cell's extensive vacuoles and not incorporated into its biochemistry. He would like to see Wolfe-Simon's team demonstrate a functional arsenic-containing enzyme, for example. Steven Benner, an astrobiologist at the Foundation for Applied Molecular Evolution in Gainesville, Florida, is more skeptical: That GFAJ-1 uses arsenic as a replacement for phosphorus, “is, in my opinion, not established by this work,” he says. 

Wolfe-Simon isn't arguing that GFAJ-1 prefers, or even naturally uses, arsenic. Mono Lake has a lot of phosphorus as well as arsenic, and the strain grows better when supplied with phosphorus. But to her and others, GFAJ-1 is proof that phosphorus-free life forms can exist and may do so somewhere on Earth. Next, Wolfe-Simon wants to collect samples from places with high arsenic but low phosphorus concentrations in hopes of finding microbes that depend solely on the former.

Wolfe-Simon speculates that organisms like GFAJ-1 could have thrived in the arsenic-laden hydrothermal vent–like environments of early Earth, where some researchers think life first arose, and that later organisms may have adapted to using phosphorus. Others say they'll refrain from such speculation until they see more evidence of GFAJ-1's taste for arsenic and understand how the DNA and other biomolecules can still function with the element incorporated. “As in this type of game changer, some people will rightly want more proof,” says microbiologist Robert Gunsalus of the University of California, Los Angeles. “There is much to do in order to firmly put this microbe on the biological map.”

Molecular Structure to Biological Function

The late Prof. Tatsuo Miyazawa was an outstanding physical chemist, who established a number of spectroscopic methods to analyse the structures of proteins, peptides and nucleotides, and used them to understand molecular functions. He developed an infrared spectroscopic method to quantitatively analyse the secondary structures, α-helices and β-strands, of proteins. He successfully utilized nuclear magnetic resonance (NMR) methods to determine the conformations of peptides and proteins, particularly with respect to the interactions with their target molecules, which served as a solid basis for the wide range of applications of NMR spectroscopy to life science research. For example, he found that physiologically active peptides are randomly flexible in solution, but assume a particular effective conformation upon binding to their functional environments, such as membranes. He also used NMR spectroscopy to quantitatively analyse the conformer equilibrium of nucleotides, and related the dynamic properties of the modified nucleosides naturally-occurring in transfer ribonucleic acids (tRNAs) to their roles in correct codon recognition in protein synthesis. Furthermore, he studied the mechanisms of protein biosynthesis systems, including tRNA and aminoacyl-tRNA synthetases. Inspired by the structural mechanism of amino acid recognition by aminoacyl-tRNA synthetases, as revealed by NMR spectroscopy, he initiated a new research area in which non-natural amino acids are site-specifically incorporated into proteins to achieve novel protein functions (alloprotein technology).

Infrared spectroscopy of polypeptides and proteins: the amide bands

Professor Tatsuo Miyazawa (Fig. 1) graduated from the University of Tokyo in 1950. He had already joined the laboratory of Prof. San-ichiro Mizushima, and studied peptide conformation by infrared spectroscopy when he was a graduate student. After he obtained his PhD in 1956, he received a Fulbright Grant to join the laboratory of Prof. Kenneth S. Pitzer, at the Department of Chemistry, University of California, Berkeley. In 1958, he joined the laboratory of Prof. Elkan R. Blout at Children’s Cancer Research Foundation, Boston, and analysed the amide bands in the infrared spectra of polypeptides. He continued vibrational spectroscopic studies at the Institute of Protein Research, Osaka University, as an associate professor (1959–64) and a professor (1964–74). He published more than 100 papers during these periods, and was recognized as an outstanding physical chemist. 

 

 

Fig. 1

Professor Miyazawa (1927–93).

Infrared spectroscopy is a useful method for studying the chain conformations of polypeptides and proteins

. Polypeptides exhibit characteristic infrared absorption bands: amides A, B and amides I–VII . It was previously shown that amide A (3,300 cm⁻¹), amide B (3,100 cm⁻¹), amide I (1,650 cm⁻¹) and amide II (1,550 cm⁻¹) are characteristic of the CONH group . Professor Miyazawa elucidated the nature of these bands in detail by analysing monosubstituted amides, including N-methylacetamide (CH₃–CONH–CH₃), and performing theoretical calculations of the vibrations. Professor Miyazawa showed quantitatively that the amide A and B bands are due to the Fermi resonance between the fundamental N–H stretching vibration and the first overtone of the amide II vibration . Professor Miyazawa also quantitatively described the nature of the amide I and II bands by a normal coordinate analysis , as well as by infrared and Raman studies of a series of monosubstituted amides 

Figure 2

shows the calculated normal modes of the amide I–IV vibrations

.

Calculated normal modes of the amide I–IV vibrations of N-methylacetamide. 

Stahmann, Mark A. Polyamino Acids, Polypeptides, And Proteins. © 1962 by the Board of Regent of the University of Wisconsin System. Reprinted courtesy of The University of Wisconsin Press.

The frequencies of the amide I and II bands reportedly differ between the α-helical conformation and the β-conformations . Professor Miyazawa theoretically analysed the vibrational interactions among peptide groups, and established an elegant relationship between the frequencies and the chain conformations , as summarized in . This method to analyse the amide I and II bands is practically useful for vibrational spectroscopic analyses of protein main-chain conformations. Therefore, these papers by Prof. Miyazawa have been extensively cited, and are cited even today, as the primary theoretical basis. For proteins, various chain conformations may co-exist, and the amide bands IV–VI observed in the 800–500 cm⁻¹ region can be used to estimate the fractions of various conformations . As for the side chain conformations, Prof. Miyazawa established the correlations of the cystine disulfide-bond conformations to the S–S and C–S stretching frequencies .

The frequencies (cm⁻¹) and relative intensities of the amide I and II bands of polypeptides in various conformations 

Since Prof. Miyazawa’s achievements, vibrational spectroscopic methods have been greatly advanced by laser Raman and Fourier transform infrared spectroscopy, etc., and are now used to analyse the conformational properties of proteins, including protein unfolding and amyloids, for which other methods, such as X-ray crystallography, are not easily applied . The idea that the conformations of biomolecules, such as proteins, can be analysed by spectroscopic approaches has led to the current stage of NMR structural biology.

NMR analyses of nucleotides, peptides and proteins

In 1971, Prof. Miyazawa moved to the Department of Biophysics and Biochemistry, at the University of Tokyo. He performed NMR spectroscopy of biological molecules with Prof. Mitsuo Tasumi, an associate professor. In 1972, a 90-MHz NMR spectrometer (Hitachi R22) was installed, and was operated in a continuous wave (CW) mode and later in a Fourier transform (FT) mode. Then, in 1976, the first FT-NMR spectrometer with a superconducting magnet in Japan (Bruker WH-270) was installed in Miyazawa’s laboratory. With these NMR spectrometers, new projects on nucleotides, peptides and small proteins were initiated.

Nucleotides and peptides in solution are flexible with respect to the conformations around single bonds, and thus multiple conformers may exist in equilibrium. By applying NMR spectroscopy, Prof. Miyazawa succeeded in the quantitative analysis of conformer equilibria of molecules with plural flexible single bonds, for the first time. For this purpose, he chose the lanthanide-ion probe method, which utilizes the paramagnetic effects of lanthanide ions, bound at the nucleotide phosphate group, on the chemical shifts and the relaxation rates . The advantage of this method is that it can yield information about the distances and orientations of the observed nuclei relative to the lanthanide ion, spanning relatively long distances (∼10 Å), or molecules as large as the nucleotide unit. By employing the lanthanide probe analyses in combination with shorter-distance analyses of vicinal spin-coupling constants and nuclear Overhauser effects (NOEs), Prof. Miyazawa successfully determined not only the structure but also the population of each conformer in a multi-conformational equilibrium , the conformation around the C3′–O3′ bond of Up exists in an equilibrium between the G⁺ (major) and G⁻ (minor) forms when the ribose moiety assumes the C2′-endo form, but the C3′–O3′ conformation is exclusively in the G^– form when the ribose moiety assumes the C3′-endo form. This concept of ‘conformational interrelations controlling RNA structures’ subsequently led to the elucidation of the functions of post-transcriptional modifications in transfer RNAs (tRNAs), as described below.

Fractional populations of local conformations of Up

Reprinted in part with permission from Biochemistry 20, 2981–2988. Copyright 1981 American Chemical Society. A boll-and-stick model of the most preferable conformation, C3′-endo-gg-G^–-g⁺, of Up is also shown.

When a small peptide, such as a biologically active peptide, is in equilibrium between the free and bound forms with phospholipid membranes, a transferred NOE  technique can be used to analyse the conformation of the peptide bound to the membranes. The technique was first applied to mastoparan-X, one of the mast cell-degranulating peptides in the venom of Vespa xanthoptera  and then to α-mating factor, a pheromone secreted by the α-type cell of Saccharomyces cerevisiae. The merit of the TRNOE method is that the NOEs, which provide structural information, of the membrane-bound peptide can be obtained by analysing the well-resolved signals of the free peptide. Figure 4 shows the conformation of membrane-bound α-mating factor, in which the N-terminal nine residues, Trp–His–Trp–Leu–Gln–Leu–Lys–Pro–Gly, are tightly bound to the membrane and the conformations of these residues are well determined. The C-terminal four residues, Gln–Pro–Met–Tyr, are left free in the aqueous phase. Since the physiological activities of peptides are correlated with the conformations of membrane-bound molecules , the TRNOE analysis of biologically active peptides is beneficial for obtaining their structure–activity relationships. This line of study on the membrane-bound peptide conformations is currently advancing, particularly by solid-state NMR methods . On the other hand, the concept of the ‘formation of the functional peptide conformation upon binding to its target’ is being applied more generally now, for example, to the intrinsically-unfolded regions of large proteins that assume the functional conformation only when complexed with their partner proteins or nucleic acids.

 

 

Conformation of membrane-bound α-mating factor 

 Reprinted in part with permission from Wakamatsu et al. 

, Wiley-Blackwell.

As for small proteins, Prof. Miyazawa measured and analysed the 270-MHz ¹H NMR spectra of snake neurotoxins, such as erabutoxins a and b (Fig. 5) 

Erabutoxins are neurotoxic proteins from sea snake venom and consist of 62 amino acids. The NMR signals from several residues were assigned and their structural characteristics were analysed. For example, it was unambiguously concluded that the imidazole ring of His7 is only protonated upon denaturation at pH 2.85, indicating that His7 is deeply buried in the interior of the protein. In this manner, Prof. Miyazawa powerfully demonstrated that the unambiguous assignment of signals to the amino acid residues in the primary sequence of the analyte protein is essential for understanding the structure and function of the protein. Since these pioneering discoveries by Prof. Miyazawa, the NMR methods have greatly advanced, e.g. multiple stable-isotope labeling techniques and two-, three- and four-dimensional NMR spectroscopy, so that the tertiary-structure determination of proteins in solution is now generally possible for relatively small proteins, from 50 to 300 residues, and still spanning towards larger ones. Moreover, the structures of functional RNA molecules can also be determined by NMR methods. 

The 270-MHz ¹H-NMR spectra of (a) erabutoxin and (b) erabutoxin b in D₂O solution at pH 5.2 and 296 K 

These proteins consist of 62 amino acid residues. The protein concentration was 4 mM. Reprinted in part with permission from Inagaki et al., Wiley-Blackwell.

Professor Miyazawa also showed that the conformation of a small ligand bound to a protein can be analysed by NMR. In the case of an inhibitor bound to Ribonuclease (RNase) T₁ , the base orientation of the bound guanine nucleotides was successfully analysed from the NOE between the H8 and H1′ protons, together with the vicinal coupling constants between the H1′ and H2′ protons. The conformation of the guanosine moiety bound to RNase T₁ was found to be C3′-endo-syn for 2′-GMP and 3′-GMP and C3′-endo-anti for 5′-GMP and guanosine 3′,5′-bis(phosphate), suggesting that there is no specific binding site for the ribose moiety of the inhibitors. A similar analysis was performed to determine the conformation of the guanine nucleotide bound to the c-Ha-ras protein These types of NMR analyses are now quite widely performed to determine the interactions of proteins with small compounds, such as inhibitors, substrates, cofactors and medicines, as well as the interactions of proteins with other biological macromolecules, such as proteins, nucleic acids and lipid membranes. The detailed structural information of the interaction, which can only be obtained by such NMR analyses, is generally useful from basic to applied studies in life science. It should be emphasized that Prof. Miyazawa’s pioneering studies still serve as the bases for the frontier of life science research. For example, the lanthanide probe method is now being applied to study the transient and dynamic processes of protein–protein and protein–nucleic acid interactions 

Protein biosynthesis mechanisms and ‘alloproteins’

Around 1980, Prof. Miyazawa started structural and functional studies on protein biosynthesis systems, including tRNA and aminoacyl-tRNA synthetase. These studies led to the in vivo production of an ‘alloprotein’, which contains unnatural amino acids, with the support of grants-in-aid for Distinguished Research (1985–88).

The codon recognition by tRNA is an important step for the fidelity in the translation of genetic information. A variety of post-transcriptionally modified nuclesides have been identified in tRNA molecules, and among them, modified uridines in the first position of the anticodon constitute one of the most diverse and complicated groups of naturally occurring, modified nucleosides. These uridine modifications were considered to be related to the properties of the tRNA in ‘wobble’ codon recognition through non-Watson–Crick base pairing 

, but there was no structural basis for the dynamic process of wobbling. Based on the above-mentioned conformational analysis of uridine nucleotides by the lanthanide probe method, the conformational characteristics of modified uridine residues in the first position of the anticodon were analysed . It was clearly demonstrated that derivatives of 5-methyl-2-thiouridine (xm⁵s²U) in the first position of the anticodon exclusively assume the C3′-endo form to recognize adenosine (but not uridine) in the third position of the codon. In contrast, derivatives of 5-hydroxyuridine (xo⁵U) assume the C2′-endo form as well as the C3′-endo form to recognize adenosine, guanosine and uridine as the third letter of the codon (Fig. 6). Accordingly, the biological significance of the modifications of U to xm⁵s²U/xo⁵U is in the regulation of the conformational rigidity/flexibility in the first position of the anticodon, to guarantee the correct and efficient translation of codons in protein biosynthesis. This is the first successful demonstration that the dynamic properties of biological molecules in conformational equilibrium are directly relevant to their biological roles. It is interesting that a similar ‘rigid’ 2-thiouridine derivative (2-thioribothymidine) is the major stabilizing factor in the tRNAs from an extremely thermophilic bacterium, Thermus thermophilus HB8 

Base pairs of xo⁵U with adenosine (a) and uridine (b) as the third letter of the codons 

.

Professor Miyazawa revealed that the modification in the first position of the anticodon is required not only for the codon recognition but also for the tRNA identity, i.e. the specific recognition by the aminoacyl-tRNA synthetase. In Escherichia coli, the major isoleucine tRNA (tRNA^Ile_major) recognizes the AUU and AUC codons, and the minor isoleucine tRNA (tRNA^Ile_minor) recognizes the AUA codon only. The tRNA^Ile_minor was purified from 10 kg of wet cells, and yielded 10 µg of the unknown modified nucleoside N⁺, located in the first position of the anticodon. The chemical structure of N⁺ was analysed by NMR and mass spectrometry, and was confirmed by chemical synthesis, as shown in Fig. 7 . N⁺ was found to be a lysine-substituted cytidine, and was named lysidine, which must recognize adenosine, but not guanosine. Surprisingly, the unmodified tRNA^Ile_minor bearing cytidine in the first position of the anticodon, which corresponds to the methionine codon AUG, showed methionine-accepting activity and reduced isoleucine-accepting activity, indicating that the codon and amino acid specificities of a tRNA are both converted by the lysidine modification Recently, the mechanisms of the lysidine biosynthetic processes were revealed by X-ray crystallography 

Structure of the nucleoside N⁺ (lysidine) in the neutral form.

In addition to tRNA recognition, aminoacyl-tRNA synthetases are also responsible for the strict discrimination of their cognate amino acid from others, which is required for the fidelity in the translation of genetic information. Isoleucyl-tRNA synthetase (IleRS) discriminates between l-isoleucine and l-valine with a very low error rate in protein biosynthesis, although the chemical structures of l-isoleucine and l-valine are similar to each other. Although IleRS is one of the largest among the 20 aminoacyl-tRNA synthetases (115 kDa), the TRNOE technique was applied with the hope of elucidating the conformations of amino acids interacting with E. coli IleRS . The TRNOE analysis revealed that the conformations of l-isoleucine and l-valine bound to IleRS are quite similar to each other, and that the hydrophobic interaction of l-isoleucine with the active site of IleRS is more significant than that of l-valine. Furthermore, the conformation of the non-protein amino acid furanomycin bound to IleRS was also analysed by TRNOE, which clarified the mechanism of furanomycin incorporation into proteins . This is an example of the usefulness of the TRNOE method for high-molecular mass systems.

In order to obtain more stable proteins for further studies, Prof. Miyazawa isolated several aminoacyl-tRNA synthetases, including IleRS and glutamyl-tRNA synthetase (GluRS), from the above-mentioned extreme thermophile, T. thermophilus HB8. The molecular mechanism underlying the strict specificity of T. thermophilus GluRS for glutamic acid tRNA (tRNA^Glu) was analysed . GluRS is one of the aminoacyl-tRNA synthetases that require the cognate tRNA for an apparently unrelated reaction, for the formation of an aminoacyl-adenylate from ATP and the cognate amino acid (the process of amino acid activation). The studies revealed that, in the absence of tRNA^Glu, GluRS binds not only the correct substrate, l-glutamate, but also incorrect amino acids, such as d-glutamate and l-aspartate. In contrast, GluRS recognizes only l-glutamate in the presence of its cognate tRNA^Glu. Thus, the cognate tRNA^Glu acts as an allosteric effector for the specific activation of l-glutamate by GluRS. Subsequently, the crystal structures of T. thermophilus GluRS by itself and in complex with its substrates, ATP, glutamate and/or tRNA^Glu, revealed the structural basis for the tRNA-dependent glutamate activation by GluRS . Presently, a large number of studies, using many kinds of aminoacyl-tRNA synthetases from T. thermophilus, have established the structural basis for the amino acid and tRNA recognition mechanisms, mainly by X-ray crystallography .

The studies described above, as well as those on elongation factor Tu (EF-Tu) , paved the way towards the engineering of protein biosynthesis systems. Professor Miyazawa started the project to produce ‘alloproteins’ with unnatural amino acids, by using the protein biosynthesis system. As previously mentioned, E. coli IleRS recognizes the non-protein amino acid furanomycin. Furthermore, furanomycin-bound isoleucine tRNA (Fur-tRNA^Ile) can form a ternary complex with EF-Tu and GTP. Subsequently, the incorporation of furanomycin into a protein was accomplished by an in vitro translation system with an E. coli S30 extract . Thus, it was demonstrated that a protein carrying non-protein amino acids, an alloprotein, can be produced by the protein biosynthesis system. During his last few years at the University of Tokyo, Prof. Miyazawa promoted the alloprotein project, and eventually successfully produced human epidermal growth factor (hEGF) bearing a non-protein amino acid, l-2-aminohexanoic acid (Ahx) or l-norleucine, by using an E. coli secretion system  Figure 8 shows the amino acid composition analysis of the produced alloprotein. [Ahx²¹]hEGF exhibited an activity comparable to that of the natural hEGF for the stimulation of cell proliferation as well as DNA synthesis. Moreover, [Ahx²¹]hEGF is resistant to inactivation through the oxidation of the single methionine residue of hEGF. 

Reverse-phase HPLC of PTC derivatives of amino acids from a hydrolysate of [Ahx²¹]hEGF 

and the chemical structures of Met and Ahx.

After he retired from the University of Tokyo, Prof. Miyazawa continued the alloprotein project at Yokohama National University from 1988. It should be emphasized that Prof. Miyazawa is the pioneer who established alloprotein technology using non-natural amino acids in protein biosynthesis. Today, a large number of engineered aminoacyl-tRNA synthetases, which were created through random and structure-based approaches, are widely used to incorporate useful non-natural amino acids into specified positions of target proteins . The idea of using the non-natural properties of amino acids to drastically improve protein functions is becoming more and more realistic, and is regarded as ‘superprotein’ technology, as Prof. Miyazawa dreamed. Quite sadly, Prof. Miyazawa passed away suddenly in 1993, soon after he moved to the Protein Engineering Research Institute in 1991 to serve as its president.

Professor Miyazawa first used infrared spectroscopy, and primarily focused his efforts on the theoretical aspects of the field of physicochemistry. He then switched the methodology to NMR spectroscopy, and subsequently to biochemistry. He did not hesitate to change the direction of his research, and acted decisively to achieve his research goals. He always encouraged the laboratory members, from staff to students, to do everything possible to achieve their goals. Prof. Miyazawa’s attitude strongly influenced all of his coworkers, and many of his former students are now active at the forefront of biochemical research, not only in structural biology but also a broad range of life sciences.

Double-Stranded DNA Quantitation

Characterization of PicoGreen Reagent and Development of a Fluorescence-Based Solution Assay for Double-Stranded DNA Quantitation

A sensitive assay for detecting double-stranded (ds) DNA in solution is described. This assay employs a new dye, PicoGreen dsDNA quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The brightness of this reagent is due to its high quantum yield (0.5, bound to ds calf thymus DNA) and large molar extinction coefficient (70,000 cm⁻¹m⁻¹). The fluorescence enhancement of this dye upon binding dsDNA is >1000-fold, with excitation and emission maxima near those of fluorescein. Unlike Hoechst 33258, PicoGreen reagent fluorescence intensity was the same upon binding to poly(dA)·poly(dT) and poly(dG)·poly(dC) homopolymers. The PicoGreen assay allowed the detection of 25 pg/ml dsDNA, surpassing the sensitivity achieved with Hoechst 33258 by 400-fold. The linear concentration range for DNA quantitation extended over four orders of magnitude—25 pg/ml to 1 μg/ml—with a single dye concentration. Assay linearity was maintained even in the presence of salts, proteins, poly(ethylene glycol), urea, chloroform, ethanol, and agarose; some ionic detergents and heparin interfered. Linear DNAs yielded slightly brighter signals than supercoiled plasmids. Finally, the assay showed greater dsDNA:RNA selectivity than Hoechst 33258 in low ionic strength buffer and better dsDNA:single-stranded DNA selectivity in 1mNaCl.

Biochemical techniques

What is Biochemistry?

Biochemistry

Biochemistry is the study of chemical processes associated with living organisms. Biochemists use concepts of biology, chemistry, physics, mathematics, microbiology, and genetics to unravel the complex puzzles of life. Biochemical techniques are used in clinical diagnosis of infectious diseases, genetic disorders, and cancer; as well as in many forms of research to improve the quality of our lives.

Nutrition

Nutrition is the science that studies the means by which we take in and utilize food. Nutritionists study the metabolism of foods, i.e. how foods are converted and used by the body. They study the need for vitamins and trace elements in all stages of human development from pre-term infants to the elderly.

Dietetics

Dietetics is the application of nutrition to diet planning for individuals, groups and populations. Dietitians are uniquely trained to advise you on food, diet and nutrition. Only a dietitian holds a professional qualification and certain positions (such as food-service directors in hospitals) are restricted to holders of this qualification.

Analysis

Citric acid Analysis


Citric acid was analysed, using pyridine–acetic anhydride method as reported by Marrier and Boulet (1958). One ml of the diluted culture filtrate along with 1.30 ml of pyridine was added in the test tube and swirled briskly. Then 5.70 ml of acetic anhydride was added in the test tube. The test tube was placed in a water bath at 32 ± 0.25 °C for 30 min. The optical density was measured on a spectrophotometer (405 nm) and citric acid contents of the sample were estimated by comparing it with standerds (run parallel, replacing 1.0 ml of the culture filtrate with distilled water).

Composition of Medium For optimization of pH for SSF of molasses using corn cobs as carrier substrate for citric acid production by A. niger
 
Substrate corn cobs
S.no Substrate (g) 10%molasses solution (ml) pH


1 5 15 3

2 5 15 4

3 5 15 5

4 5 15 6

5 5 15 7


Composition of mdium for optimization of inoculum size for citric acid production by A. niger in SSF of molasses medium.
 
Substrate corn cobs
 
S.no Substrate (g) 10%molasses solution (ml) pH Inoculum size


(ml)

1             5          15             6                                 3

2 5 15 6 4

3 5 15 6 5

4 5 15 6 6

5 5 15 6 7

Composition of medium for optimization of temperature


Composition of medium for optimization of nitrogen sources

Substrate corn cobs


S.no

Nitrogen Sources w/w 1%

1 Ammonium sulphate

2 Corn steep liqour

3 Urea

4 Peptone

5 Yeast extract


PH, 6


Inoculum size, 5ml.

Temperature, 35oC.









Substrate corn cobs

S.no pH Inoculum size (ml) Temprature oC








1                 6              3           25

2              6            4                30

3 6 5 35

4 6 6 40

5 6 7 45